Publication

Stabilization of cyclohexanone monooxygenase by computational and experimental library design

Fürst, M., Boonstra, M., Bandstra, S. & Fraaije, M., Sep-2019, In : Biotechnology and Bioengineering. 116, 9, p. 2167-2177 11 p.

Research output: Contribution to journalArticleAcademicpeer-review

Enzymes often by far exceed the activity, selectivity and sustainability achieved with chemical catalysts. One of the main reasons for the lack of biocatalysis in the chemical industry is the poor stability exhibited by many enzymes when exposed to process conditions. This dilemma is exemplified in the usually very temperature‐sensitive enzymes catalyzing the Baeyer‐Villiger reaction (BVMOs), which display excellent stereo‐ and regioselectivity and offer a green alternative to the commonly employed, explosive peracids. Here we describe a protein engineering approach applied to cyclohexanone monooxygenase from Rhodococcus sp. HI‐31, a substrate promiscuous enzyme that efficiently catalyzes the production of the nylon‐6 precursor ε‐caprolactone. We employed a framework for rapid enzyme stabilization by computational libraries (FRESCO), which predicts protein‐stabilizing mutations. From 128 screened point mutants, approximately half had a stabilizing effect, albeit mostly to a small degree. To overcome incompatibility effects observed upon combining the best hits, an easy shuffled library design strategy was devised. The most stable and highly active mutant displayed an increase in unfolding temperature of 13 °C, and an approximately 33x increase in half‐life at 30 °C. In contrast to the wild‐type enzyme, this thermostable 8x mutant is an attractive biocatalyst for biotechnological applications.
Original languageEnglish
Pages (from-to)2167-2177
Number of pages11
JournalBiotechnology and Bioengineering
Volume116
Issue number9
Early online date23-May-2019
Publication statusPublished - Sep-2019

    Keywords

  • Baeyer–Villiger monooxygenase, computational design, cyclohexanone, stabilization, thermostability, BAEYER-VILLIGER-MONOOXYGENASES, CRYSTAL-STRUCTURE, PHENYLACETONE MONOOXYGENASE, MUTATIONS, STABILITY, CATALYST, ALCOHOL, ENZYMES

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